The transforming growth factor (TGF) superfamily of signaling molecules fulfills essential functions in development and disease. Activins are members of this superfamily. Activin A (Act A) and TGF overlap in their intracellular signaling cascades and function and have multiple common downstream targets. We have previously demonstrated that coordinated loss of TGF receptor II and E-cadherin results in cell invasion. These genetically manipulated esophageal keratinocytes show activation of the common downstream target Smad2 despite the absence of functional TGF receptor II. These data indicate that Act A may be responsible for Smad2 activation in the absence of canonical TGF signaling in esophageal keratinocytes. The interaction of the epithelium with the surrounding microenvironment is essential for maintaining epithelial homeostasis. A knowledge gap exists as no mechanism for the dual function of Act A in the maintenance and disruption of homeostasis similar to TGF has been described yet. Our long-term goal is to produce mechanistic insights into the signaling pathways mediating epithelial-mesenchymal crosstalk. Our central hypothesis for this study is that Act A signaling is a major regulator of epithelial-mesenchymal transition (EMT) and epithelial-mesenchymal crosstalk in the esophagus. Our preliminary data show the increased secretion of Act A in organotypic cultures and increased cell invasion of esophageal keratinocytes in Boyden chamber assays after Act A stimulation. Therefore, we propose to utilize state-of-the-art approaches and novel mouse models to demonstrate the role of Act A in squamous homeostasis in vitro and in vivo. These data will advance our understanding of TGF superfamily signaling in the esophagus. To test our hypothesis we pursue the following specific aims: 1) To determine how Act A signaling results in EMT and esophageal cell invasion. 1a) To define autocrine Act A signaling in esophageal epithelial cells and 1b) To analyze the functional consequences of ActRIIB loss. 2) To determine how paracrine Act A signaling induces fibroblast activation. 3) To identify the role of Act A in esophageal homeostasis in vivo. In the first aim, an established shRNA approach to suppress Act A and ActRIIB, as well as overexpression thereof will be used to show the causal effects of Activin signaling on EMT and cell invasion. We have shown the successful use of organotypic cultures to demonstrate the disruption of epithelial homeostasis by altered epithelial-mesenchymal crosstalk. We will utilize this system to identify how paracrine Act A secretion induces fibroblast activation in the second aim. In specific aim 3, we make use of mouse models with targeted loss of E-cadherin and TGF receptor in the esophagus, which we have successfully developed. The approach is innovative, because it mimics the microenvironment by incorporating stromal and epithelial components in the analysis. The proposed research is significant, because it is expected to identify the role of a novel player, Activin A, in esophageal homeostasis.